Discovery and validation of cancer biomarkers using a protein analysis methodology to analyze specimens

ABSTRACT

Methods are provided for the analysis, including the serial analysis, of very small samples of tissue. The methods utilize a nanofluidic proteomic immunoassay (NIA) to quantify total and low-abundance protein isoforms in a small amount of lysate. NIA detection accurately measure oncoprotein expression and activation in limited clinical specimens, including isoforms that differ in post-translational modifications, such as phosphorylation, and the like. The NIA detection method combines isoelectric protein focusing and antibody detection in a nanofluidic system.

BACKGROUND OF THE INVENTION

The recent explosion of information in the fields of genomics andproteomics has provided a rich ground for the discovery of moleculartargets against which therapeutic and/or diagnostic agents can bedirected. Tissues for potential target discovery may include tumors andother malignant growths, or infected or inflamed tissues. For example,methods have been described for gene expression profiling of tumor cells(see any one of Ono et al. (2000) Cancer Res. 60(18):5007-11; Svaren etal. (2000) J Biol Chem.; or Forozan et al. (2000) Cancer Res.60(16):4519-25 for examples). Similarly, proteomics has been used toprofile the protein expression in tumor samples (see Minowa et al.(2000) Electrophoresis 21(9):1782-6; Cole et al. (2000) Electrophoresis21(9):1772-81; Simpson et al. (2000) Electrophoresis 21(9):1707-32);etc.

Cancer is caused by multiple genetic events that result in theactivation of proto-oncogenes and/or the inactivation of tumorsuppressor genes. In some areas of the world, cancer has become orshortly will become the leading disease-related cause of death of thehuman population. For example, in the United States, cancer is thesecond leading cause of death behind cardiovascular disease, and it isprojected that cancer will become the leading cause of death within afew years. The medical treatment of cancer still has many unmet needs.Surgery and radiation are generally only successful if the cancer isfound at an early, localized stage. Once the disease has progressed tolocally advanced cancer or metastatic disease, these therapies are lesssuccessful. Existing chemotherapeutic treatments are largely palliativein these advanced tumors, particularly in the case of the commonepithelial tumors such as lung, colorectal, breast, prostate, andpancreatic cancers. Although a few chemotherapeutic regimens haveyielded lasting remissions or cures (for example, in testicular cancerand childhood leukemias), it is clear that new therapeutic options arenecessary.

The transformation and malignant growth of tumor cells is a complexprocess, which can be variable even within a particular tissue type.Analytical methods that can define the phenotype of tumor cells areuseful in determining appropriate therapy, and are therefore of clinicalinterest. Additionally, knowledge of the mechanism by which achemotherapeutic agent acts is useful determining optimal formulationand dosage of such agents; in screening for agents effective in treatingcancer; and in following patients through a course of treatment.

A variety of post translational modifications of proteins take place.These modifications include phosphorylation, glycosylation, prenylation,and the like. The modifications, particularly reversiblephosphorylation, can be a molecular mechanism by which intracellularsignals are transmitted. A substantial number of signaling proteins arekinases or phosphatases that act on serine, threonine, and tyrosineresidues. Wth over 2000 human genes predicted to code for kinases andthe potential for each kinase to act on multiple targets, signalingnetworks are immensely complex. An important step towards unravelingthis complexity is the development of new proteomics technologies thatcan quantitatively monitor the phosphorylation states of signalingproteins in a multiplex fashion. Such technologies would enable thedetailed analysis of signaling pathways in a global perspective and therapid identification of previously unrecognized signaling events.

Phosphorylation of target proteins by kinases is an important mechanismin signal transduction and for regulating enzyme activity. Tyrosinekinases (TK) are a class of over 100 distinct enzymes that transfer aphosphate group from ATP to a tyrosine residue in a polypeptide (Table1). Tyrosine kinases phosphorylate signaling, adaptor, enzyme and otherpolypeptides, causing such polypeptides to transmit signals to activate(or inactive) specific cellular functions and responses. There are twomajor subtypes of tyrosine kinases, receptor tyrosine kinases andcytoplasmic/non-receptor tyrosine kinases.

To date there have been approximately 60 receptor tyrosine kinases(RTKs; also known as tyrosine receptor kinases (TRK)) described inhumans. These kinases are high affinity receptors for hormones, growthfactors and cytokines (Robinson et al. (2001) Oncogene 19:5548-57). Thebinding of hormones, growth factors and/or cytokines generally activatesthese kinases to promote cell growth and division. Exemplary kinasesinclude insulin-like growth factor receptor, epidermal growth factorreceptor, platelet-derived growth factor receptor, etc. Most receptortyrosine kinases are single subunit receptors but some, for example theinsulin receptor, are multimeric complexes. Each monomer contains anextracellular N-terminal region, a single transmembrane spanning domainof 25-38 amino acids, and a C-terminal intracellular domain. Theextracellular N-terminal region is composed of a very large proteindomain which binds to extracellular ligands e.g. a particular growthfactor or hormone. The C-terminal intracellular region provides thekinase activity of these receptors. Receptor tyrosine kinases are keyregulators of normal cellular processes and play a critical role in thedevelopment and progression of many types of cancer (Zwick et al. (2001)Endocr. Relat. Cancer 8:161-173).

Cancer is frequently associated with the abnormal expression andphosphorylation of oncogenes. Specific tumors may be characterized bythe discrete activation of specific oncogenes such as MYC, BCL2(encoding B cell lymphoma protein-2) and BCR-ABL. Targeted inactivationof oncoproteins is emerging as a specific and effective therapy forcancer. The best known example of a targeted therapy is imatinibmesylate, a small molecule that inactivates several tyrosine kinases,including the BCR-ABL tyrosine kinase in CML. Imatinib treatment resultsin tumor cell signaling changes in vitro, leading to cell death. Ingeneral, the ability to detect specific oncoproteins and theiractivation state is likely to be highly useful toward the development ofnew therapeutics as well as in monitoring the effectiveness of thesetreatments and in evaluating apparent therapeutic resistance_(13,21).

Current methods of protein detection are insensitive to detecting subtlechanges in oncoprotein activation that underlie key cancer signalingprocesses. The requirement for large numbers of cells precludes serialtumor sampling for assessing a response to therapeutics. The presentinvention addresses this need.

SUMMARY OF THE INVENTION

Methods are provided for the analysis, including the serial analysis, ofvery small amounts of clinical specimens. Samples of interest includehuman tissue, particularly cancer and other lesions, e.g. blood or solidtumor microbiopsy samples such as fine needle aspirate. Samples may betaken at a single timepoint, or may be taken at multiple timepoints.Samples may be as small as 100,000 cells, as small as 5000 cells, assmall as 1000 cells, as small as 100 cells or less.

The methods utilize a nanofluidic proteomic immunoassay (NIA) toquantify total and low-abundance protein isoforms in a small amount oflysate. NIA detection accurately measure oncoprotein expression andactivation in limited clinical specimens, including isoforms that differin post-translational modifications, such as phosphorylation, and thelike. The NIA detection method combines isoelectric protein focusing andantibody detection in a nanofluidic system. In some embodiments of theinvention, the NIA detection is performed on a sample that has beenfrozen, where the cells are lysed after thawing. Blood cells may beretained in the sample to reduce variability. Analysis may be performedfor up to 60 minutes following sample obtainment, provided the samplesare maintained on ice.

In some embodiments of the invention, protein isoform biomarkers areutilized to determine a characteristic of a tumor, includingresponsiveness to drug treatment. In some embodiments, detection of thepresence of the single phosphorylated ERK2 isoform is indicative ofresponsiveness to tyrosine kinase inhibitors by the cancer, e.g. bychronic myelogenous leukemia cells (CML). The isoform may be detected byNIA, or by conventional methods. In some embodiments of the invention,samples are analyzed by NIA for a 23 member panel of parameterscomprising normalized values for: total ERK1/2, phospho-ERK1/2,unphosphorylated ERK1/2, total ERK1, phospho=ERK1, pERK1, ppERK1,unphosphorylated ERK1, total ERK2, phospho-ERK2, pERK1=2, ppERK2,unphosphorylated ERK2; and ERK relative ratios for: % phospho-ERK1/2, %unphosphorylated ERK1/2, % phospho-ERK1, % unphosphorylated ERK1, %phospho-ERK2, % unphosphorylated ERK2, % pERK1, % ppERK1, % pERK2, and %ppERK2.

In some embodiments of the invention, the NIA methods are used tomeasure changes in cancer cell protein isoforms over time, in responseto treatment, etc. Because the methods of the invention only needminimal amounts of specimen, the invention has reduced to practice aminimally invasive protocol for obtaining serial protein profiles afterinitiating treatment, allowing the determination of predictive proteinbiomarkers by quantifying early changes in protein activity in patientsstarting treatment. Analysis of protein change over time in a patientlesion is a better biomarker than any single protein measurement.

In some embodiments, precise measurements of MYC and BCL2 proteins byNIA provide a means of distinguishing lymphoma from normal tissue.

The methods of the invention are of interest for determining patterns ofmodifications, and the like that define disease states or classifysubsets of disease (including staging and subsets of cancers, autoimmunediseases, and the like); that follow response to therapy; that determineresponse patterns after exposure to a specific agent, and the like. Thisinformation is useful in the development of therapies, as a diseaseprognostic, determining patient specific therapies, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1A-1E. NIA for the quantitative analysis of oncoproteins. (FIG. 1A)Schematic of the use of NIA for the measurement of oncoproteins fromclinical specimens. NIA can be used to measure oncoprotein expressionand phosphorylation in clinical specimens, incorporating charge basedseparation coupled to antibody detection. (FIG. 1B, FIG. 1C) Detectionof recombinant MYC (FIG. 1B) or BCL2 (FIG. 1C) oncoproteins in vitro byNIA. Left, representative NIA traces of MYC (black, 0.004 pg nl⁻¹; red,0.012 pg nl⁻¹; blue, 0.04 pg nl⁻¹; green, 0.12 pg nl⁻¹) or BCL2 (green,0.0019 pg nl⁻¹; red, 0.0075 pg nl⁻¹; blue, 0.03 pg nl⁻¹; orange, 0.12 pgnl⁻¹; purple, 0.47 pg nl⁻¹; black, 1.8 pg nl⁻¹). Right, the data arerepresented as the normalized peak area versus protein concentration.The calculated correlation coefficients are shown. (FIG. 1D, FIG. 1E)Detection of Myc (FIG. 1D) or Bcl2 (FIG. 1E) oncoproteins in transgenicmouse tumors obtained from serial FNAs by NIA. Serial FNAs were obtainedfrom subcutaneous tumors in mice before (on) and after (off) thesuppression of expression of Myc or Bcl2. Right, corresponding westernblot analysis. Data are represented as peak areas detected by NIA; meanof three replicates per sample±s.e.m.

FIG. 2A-2B. NIA for the detection of oncoproteins in human cancerspecimens. (FIG. 2A) Left, representative traces of NIA performed on sixclinical biopsy specimens for MYC, BCL2 and HSP-70. Middle, bar graphsshowing peak areas detected by NIA; mean of four replicates persample±s.e.m. Right, western blot analysis of the specimens. (FIG. 2B)Normalized NIA data from a prospective analysis of MYC (top) and BCL2(bottom) oncoprotein expression in two sets of clinical specimens. Set 1included eight Burkitt's lymphoma, nine follicular lymphoma, and tennormal samples (the first four normal specimens were benign lymph nodesand the last six normal specimens were control peripheral mononuclearcell samples). Set 2 included seven Burkitt's lymphoma, elevenfollicular lymphoma, five marginal zone (MZ) lymphoma and nine DLBCLspecimens. Data are represented as the normalized peak areas detected byNIA; mean of four replicates per sample±s.e.m.

FIG. 3. NIA detection of changes in oncoprotein activation in CML cellstreated with imatinib. NIA was used to quantify proteomic changes in theK562 cell line treated with imatinib in vitro for 0 h, 4 h and 20 h.MEK1, MEK2, phospho-JNK (pJNK), pSTAT5 and activated caspase-3 weredetected by NIA. From left to right: representative traces for theprotein of interest, bar graph of NIA quantification of each protein,NIA pseudoblot representation and western blot data. Peaks on the tracesthat represent phosphorylated isoforms are indicated with black arrows.All measurements were performed in six replicates, and bar graph dataare represented as the mean peak area±s.e.m.

FIG. 4A-4C. NIA detected changes in pERK in individuals with CML whoresponded to imatinib treatment. (FIG. 4A) Representative NIA traces oftotal ERK for a subject who responded to treatment. Traces beforeinitiating treatment (left) and during treatment (middle) are shown. Theblue box highlights a specific change in the abundance of pERK2 (peak3). Right, similar results obtained by FACS analysis of clinicalspecimens. The black box highlights pERK changes detected by FACS. PE,phycoerythrin. (FIG. 4B) Representative NIA traces of total ERK for asubject who failed to respond to treatment, analyzed as in a. (FIG. 4C)Quantification of NIA pERK2 peak 3 analysis of eight subjects before andafter initiating treatment. Results are represented as the percentage ofpERK2 peak 3 divided by the sum of total phosphorylated andunphosphorylated ERK peaks. Experiments were performed in triplicate.

FIG. 5A-5C. NIA detected decrease in oncoproteins upon treatment withbiologic response modifying therapeutic agent. (FIG. 5A) Schematic forthe use of NIA to assess proteomic changes in clinical tumor specimens.Patients undergo pretreatment tumor sampling at baseline and again after8 d of treatment with atorvastatin. (FIG. 5B) NIA analysis of tumorcells for changes in pSTAT3 and pSTAT5. (FIG. 5C) NIA quantification ofpSTAT3 and pSTAT5±s.e.m. Samples were run in triplicate.

FIG. 6A-6B. Sensitivity of NIA versus Western Blot for MYC and BCL2 inCellular Lysates. (FIG. 6A) The measurement of oncoprotein expression intransgenic mouse tumors. The Tet-off system was used to generateconditional transgenic mouse models of MYC or BCL2 inducedlymphomagenesis. Using tumor derived cell lines from these transgenicmodels, we were able to conditionally regulate the levels of either MYCor BCL2 oncoprotein expression by titrating the concentration ofdoxycycline (dox) in their growth media. Representative NIA tracings areshown (green=dox 0 ng/ml, blue=0.01 ng/ml, red=0.05 ng/ml, black=2ng/ml, orange=20 ng/ml). (FIG. 6B) NIA peak height and western blotdensitometer quantification are graphed. Corresponding analysis byWestern blots is shown. Results obtained were statistically significantfor MYC (Pearson correlation R=0.99) and BCL2 (Pearson correlationR=0.94).

FIG. 7A-7D. NIA Analysis of ERK and MEK1 Isoforms in a Panel of 27Lymphoma and Control Specimens. NIA was used to measure phosphorylatedand unphosphorylated isoforms of ERK and MEK1 in clinical patientspecimens. (FIG. 7A, FIG. 7C) Left: Representative NIA traces of totalERK and total MEK1 (phosphorylated isoforms are more cationic, blackarrows. “p”. Unphosphorylated isoforms are labeled “u” in green).Center: NIA quantification for total ERK and total MEK1. Right: Westernblot for total ERK and phosphorylated MEK1 in representative Burkitt'slymphoma, follicular lymphoma, and benign lymph tissues are shown. (FIG.7B, FIG. 7D) Normalized NIA quantitation. NIA pERK and pMEK1 values werenormalized against HSP70 values for each of 27 samples (left). PercentpERK and pMEK1 were calculated and graphed (right). All data isrepresented as the mean of four replicates per sample+/−standard error.

FIG. 8. NIA Peak Identification of MEK1/2 Isoforms. Antibodies specificfor total MEK1 (green traces), total MEK2 (black traces), pMEK1 (bluetraces) and pMEK2 (red traces) were used to probe K562 cell line usingNIA.

FIG. 9. NIA Analysis of STAT3 Phosphorylation Following ImatinibTreatment in vitro. pSTAT3 was measured by NIA or FACS in K562 cellstreated with imatinib or PBS for 0, 4, 8, 12, 16, 20 or 24 h in vitro.

FIG. 10A-10C. NIA Identifies a Decrease in a Specific Phospho-ERKIsoform upon Imatinib Treatment in vitro and in vivo. (FIG. 10A) NIAdetected a change in phospho-ERK in vitro in the K562 CML cell lineafter treatment with 24 hours with imatinib (peak 3, blue box). pERK wasanalyzed by Western blot and FACS (FIG. 10B) Peripheral blood tumorcells were isolated from a CML patient before and after initiatingimatinib treatment. NIA demonstrates the eradication of pERK2 peak 3 invivo in a CML patient treated with imatinib. Representative NIA tracesobtained in triplicate are shown. (FIG. 10C) NIA Analysis of Totalversus Phospho-ERK in CML Patients Treated with Tyrosine KinaseInhibitors in vivo. Phospho-ERK was measured by NIA using an antibodythat detects total (black trace) versus phospho-specific ERK antibody(blue trace). Phospho-ERK2 peak 3 is highlighted by the box.

FIG. 11. Comparison of Basal pERK2 in Patients with CML. Basal p-ERK2values are graphed for 7 patients that subsequently responded to TKI(“responded to TKI”), and 5 patients that subsequently did not respondto TKI treatment (“refractory”). There is no statistically significantdifference between the mean values for each group (unpaired t-test, 2tailed, p=0.1678). Mean value for each patient+/−SEM of experimentsperformed in triplicate are graphed.

FIG. 12. Table of best responses.

FIG. 13A-13B. NIA of total ERK in resected carcinoma.

FIG. 14A-14B. FIG. 14A Freezing the tumor prior to NIA analysis isequivalent to immediate processing and analysis. Representative data ofseven ERK isoforms in an FNA lysed immediated after sample collection(fresh) versus a parallel aliquot of the same FNA lysed after storage at−80 degrees C. FIG. 14B Transport time of up to 60 minutes on ice doesnot affect NIA results. Representative data of ERK and AKT isoforms inFNA kept on ice for 0, 30, or 60 minutes. NIA assays were performed intriplicate.

FIG. 15A-15B. FNAs from 20 tumors were analyzed using NIA to measurepercent ERK phosphorylation. Samples were run in triplicate. FIG. 15AMean percent phosphorylation for each FNA is graphed, +/−SD. FIG. 15BGraph of coefficient of variance (SD/mean) for replicates of each FNA.Each bar is a ratio of: the value of % phospho-ERK for the non-tumorspecimen divided by the value of the % phospho-ERK for the paired tumorspecimen. The Y-axis of the graph is on a log scale.

FIG. 16A-16B. FIG. 16A % phospho-Erk is a specific marker for kidneycancer. As shown in the graph, each bar is a ratio of: the value of %phospho-ERK for the non-tumor kidney specimen divided by the value ofthe % phospho-ERK for the paired kidney tumor specimen. The Y-axis ofthe graph is on a log scale. FIG. 16B % ppERK1 is a specific marker forhead and neck cancer.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Methods are provided for nanofluidic proteomic immunoassay (NIA),including the serial analysis of tissues including cancer and otherlesions, e.g. solid tumor microbiopsy samples, lesions involvinginflammatory conditions, e.g. MS lesions, synovial fluid of rheumatoidarthritis patients, pancreatic cell samples from IDDM patients, and thelike. Samples may be taken at a single timepoint, or may be taken atmultiple timepoints. Samples may be as small as 100,000 cells, as smallas 5000 cells, as small as 1000 cells, as small as 100 cells or less.NIA detection accurately measure oncoprotein expression and activationin limited clinical specimens, including isoforms that differ inpost-translational modifications, such as phosphorylation, and the like.The NIA detection method combines isoelectric protein focusing andantibody detection in a nanofluidic system.

Biopsy samples can be maintained on ice for more than 30 minutes, ormore than 60 minutes, usually not more than about 120 minutes followingobtention from a patient. The sample is usually maintained without lysisof cells or red blood cells. The sample, or a lysate thereof, is stablewhen stored frozen at −80 degrees C. for long periods of time. Thus insome embodiments of the invention the analysis is performed on apreviously frozen sample.

In some embodiments of the invention, protein isoform biomarkers areutilized to determine a characteristic of a tumor, includingresponsiveness to drug treatment. Proteins of interest as biomarkersinclude, without limitation:

Tran- DNA Cell Cycle scription Apoptosis Signaling Repair Cyclin D1, 2,4 MYC p53, MDM2 RAS ATM P15, 16, 19 STAT3/5 BCL2, BCLxl MAPK, ERK ATRRB, 107 FOS, JUN Caspases PI3K, MEK, AKT H2AX E2F1, 2, 3, 4 TWIST TRAILJNKs MRE11 P21 NFκB FAD, FAS CRKL, JAKs RAD51 FoxO GSK3β DNAPK BIM, BAXBCR, TCR ABL

Protein isoform biomarkers may be utilized to determine a characteristicof a tumor, including responsiveness to drug treatment. In someembodiments, detection of the presence of the single phosphorylated ERK2isoform is indicative of responsiveness to tyrosine kinase inhibitors bythe cancer, e.g. by chronic myelogenous leukemia cells (CML). Theisoform may be detected by NIA, or by conventional methods if sample isnot limiting.

In some embodiments of the invention, samples are analyzed by NIA for a23 member panel of parameters comprising normalized values for: totalERK1/2, phospho-ERK1/2, unphosphorylated ERK1/2, total ERK1,phosphor=ERK1, pERK1, ppERK1, unphosphorylated ERK1, total ERK2,phospho-ERK2, pERK1=2, ppERK2, unphosphorylated ERK2; and ERK relativeratios for: % phospho-ERK1/2, % unphosphorylated ERK1/2, % phospho-ERK1,% unphosphorylated ERK1, % phospho-ERK2, % unphosphorylated ERK2, %pERK1, % ppERK1, % pERK2, and % ppERK2. In one embodiment, an initialanalysis generates a comprehensive profile of all 23 parameters. Thedataset is then analyzed to determine the parameter or parameters thatbest identifies a tumor of interest, such that in sequent tumor analysisa refined parameter set is chosen.

For relative ratio measurements, a single, pan-specific antibody thatrecognizes all isoforms of the protein may be used, for examplepan-specific ERK antibody, etc. The total amount of the protein, e.g.ERK2, Erk1, etc. is determined, and NIA is used to calculate the percentthat is phosphosphorylated. NIA generates peaks, and the area of eachpeak was calculated by dropping verticals to the baseline at the peakstart and end, and summing the area between the start and endpoints. NIAhas been shown to be able to discriminate between and quantitatephosphorylated and unphosphorylated isoforms of ERK in a single sample,using a total ERK antibody.

For normalized value measurements a similar process is used, but inaddition the assay utilizes an antibody for the protein of interest,e.g. pan-specific ERK antibody, and a loading control antibody, e.g.HSP-70 antibody, and the like, for normalization. NIA is utilized todiscriminate the different isoforms.

In some embodiments % phospho-ERK is used as a specific biomarker forkidney cancer, for example as shown in FIG. 16A. In other embodiments, %ppERK1 is used as a specific biomarker for head and neck cancer. Thecontrol tissue for head and neck cancer will be selected based on thesite of the head and neck tumor. For example, salivary gland tumor ispaired with the same patient's normal salivary gland. Tongue tumor ispaired with the same patient's normal tongue tissue, see FIG. 16B.

Comparisons may be performed between tissue suspected of being a tumortissue and a paired normal or on-tumor control tissue, e.g. a suspectedmelanoma or basal cell carcinoma sample, v. an adjacent non-tumor skinsample, and the like. Comparisons may also be performed with referencetumor tissue, with a time series of samples, e.g. before and aftertreatment, and the like. A ratio may be non-tumor/tumor, ortumor/non-tumor. In some embodiments a ratio provides a more predictiveor diagnostic biomarker than a single measurement of tumor or normal.

Mammalian species that provide tissue for analysis include canines;felines; equines; bovines; ovines; etc. and primates, particularlyhumans. Animal models, particularly small mammals, e.g. murine,lagomorpha, etc. may be used for experimental investigations. Animalmodels of interest include those for models of tumors, immuneresponsiveness, and the like.

Regions and/or time points of interest for screening are obtained byneedle biopsy or equivalent techniques. The cells are lysed, and thesample proteins are resolved by IEF in a short length of capillary.Resolved proteins are then captured to the capillary wall byphotochemically activated molecules lining the capillary. Suchimmobilization of the proteins allows immune complexes to be formedafter the separation step, as a means of specific detection of targetproteins. Because the protein-antibody complexes are immobilized in thecapillary, chemiluminescence reagents can be flowed through thecapillary, and light from the entire capillary can be imaged onto a CCDcamera. See, for example, O'Neill et al. (2006) PNAS 103:15153-16158,herein specifically incorporated by reference.

In one embodiment of the invention, the NIA is used to guide selectionof patient appropriate agents for therapy. A particular advantage of theinvention is the ability to provide individualized diagnosis, takingadvantage of small sample size to assess cancer patterns of expressionover time.

The information obtained from NIA is used to monitor treatment, modifytherapeutic regimens, and to further optimize the selection oftherapeutic agents. Wth this approach, therapeutic and/or diagnosticregimens can be individualized and tailored according to the dataobtained at different times over the course of treatment.

Definitions

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, animal species or genera,and reagents described, as such may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by the appendedclaims.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a compound” includes a plurality of suchcompounds and reference to “the agent” includes reference to one or moreagents and equivalents thereof known to those skilled in the art, and soforth. All technical and scientific terms used herein have the samemeaning as commonly understood to one of ordinary skill in the art towhich this invention belongs unless clearly indicated otherwise.

Post-Translational Modification

Glycosylation.

Among the post-translational modifications that can be probed, areprotein specific glycoslyation. Membrane associated carbohydrate isexclusively in the form of oliogsaccharides covalently attached toproteins forming glycoproteins, and to a lesser extent covalentlyattached to lipid forming the glycolipids. Glycoproteins consist ofproteins covalently linked to carbohydrate. The predominant sugars foundin glycoproteins are glucose, galactose, mannose, fucose, GalNAc, GlcNAcand NANA. The distinction between proteoglycans and glycoproteinsresides in the level and types of carbohydrate modification. Thecarbohydrate modifications found in glycoproteins are rarely complex:carbohydrates are linked to the protein component through eitherO-glycosidic or N-glycosidic bonds. The N-glycosidic linkage is throughthe amide group of asparagine. The O-glycosidic linkage is to thehydroxyl of serine, threonine or hydroxylysine. The linkage ofcarbohydrate to hydroxylysine is generally found only in the collagens.The linkage of carbohydrate to 5-hydroxylysine is either the singlesugar galactose or the disaccharide glucosylgalactose. In ser- andthr-type O-linked glycoproteins, the carbohydrate directly attached tothe protein is GalNAc. In N-linked glycoproteins, it is GlcNAc.

The predominant carbohydrate attachment in glycoproteins of mammaliancells is via N-glycosidic linkage. N-linked glycoproteins all contain acommon core of carbohydrate attached to the polypeptide. This coreconsists of three mannose residues and two GlcNAc. A variety of othersugars are attached to this core and comprise three major N-linkedfamilies: High-mannose type contains all mannose outside the core invarying amounts; hybrid type contains various sugars and amino sugars;complex type is similar to the hybrid type, but in addition, containssialic acids to varying degrees.

Acylation.

Many proteins are modified at their N-termini following synthesis. Inmost cases the initiator methionine is hydrolyzed and an acetyl group isadded to the new N-terminal amino acid. Some proteins have the 14 carbonmyristoyl group added to their N-termini. The donor for thismodification is myristoyl-CoA. This latter modification allowsassociation of the modified protein with membranes. For example, thecatalytic subunit of cyclicAMP-dependent protein kinase (PKA) ismyristoylated.

Methylation.

Post-translational methylation occurs at lysine residues in someproteins such as calmodulin and cytochrome c. The activated methyl donoris S-adenosylmethionine.

Phosphorylation.

Post-translational phosphorylation is one of the most common proteinmodifications that occurs in animal cells. The vast majority ofphosphorylations occur as a mechanism to regulate the biologicalactivity of a protein and as such are transient. In animal cells serine,threonine and tyrosine are the amino acids subject to phosphorylation.The largest group of kinases are those that phosphorylate either serinesor threonines and as such are termed serine/threonine kinases. The ratioof phosphorylation of the three different amino acids is approximately1000/100/1 for serine/threonine/tyrosine. Although the level of tyrosinephosphorylation is minor, the importance of phosphorylation of thisamino acid is profound. As an example, the activity of numerous growthfactor receptors is controlled by tyrosine phosphorylation.

Sulfation.

Sulfate modification of proteins occurs at tyrosine residues such as infibrinogen and in some secreted proteins, e.g. gastrin. The universalsulfate donor is 3′-phosphoadenosyl-5′-phosphosulphate (PAPS).

Prenylation.

Prenylation refers to the addition of the 15 carbon farnesyl group orthe 20 carbon geranylgeranyl group to acceptor proteins, both of whichare isoprenoid compounds derived from the cholesterol biosyntheticpathway. The isoprenoid groups are attached to cysteine residues at thecarboxy terminus of proteins in a thioether linkage (C-S-C). A commonconsensus sequence at the C-terminus of prenylated proteins has beenidentified and is composed of CAAX, where C is cysteine, A is anyaliphatic amino acid (except alanine) and X is the C-terminal aminoacid. In order for the prenylation reaction to occur the threeC-terminal amino acids (AAX) are first removed and the cysteineactivated by methylation in a reaction utilizing S-adenosylmethionine asthe methyl donor. Important examples of prenylated proteins include theoncogenic GTP-binding and hydrolyzing protein Ras and the g-subunit ofthe visual protein transducin, both of which are farnesylated. NumerousGTP-binding and hydrolyzing proteins (termed G-proteins) of signaltransduction cascades have g-subunits modified by geranylgeranylation.

Vitamin C-Dependent Modifications.

Modifications of proteins that depend upon vitamin C as a cofactorinclude proline and lysine hydroxylations and carboxy terminalamidation. The hydroxylating enzymes are identified as prolylhydroxylase and lysyl hydroxylase. The donor of the amide for C-terminalamidation is glycine. The most important hydroxylated proteins are thecollagens. Several peptide hormones such as oxytocin and vasopressinhave C-terminal amidation.

Vitamin K-Dependent Modifications.

Vitamin K is a cofactor in the carboxylation of glutamic acid residues.The result of this type of reaction is the formation of aγ-carboxyglutamate (gamma-carboxyglutamate), referred to as a glaresidue. The formation of gla residues within several proteins of theblood clotting cascade is critical for their normal function. Thepresence of gla residues allows the protein to chelate calcium ions andthereby render an altered conformation and biological activity to theprotein. The coumarin-based anticoagulants, warfarin and dicumarolfunction by inhibiting the carboxylation reaction.

Selenoproteins.

Selenium is a trace element and is found as a component of severalprokaryotic and eukaryotic enzymes that are involved in redox reactions.The selenium in these selenoproteins is incorporated as a unique aminoacid, selenocysteine, during translation. A particularly importanteukaryotic selenoenzyme is glutathione peroxidase. This enzyme isrequired during the oxidation of glutathione by hydrogen peroxide (H₂O₂)and organic hydroperoxides. Incorporation of selenocysteine by thetranslational machinery occurs via an interesting and unique mechanism.The tRNA for selenocysteine is charged with serine and thenenzymatically selenylated to produce the selenocysteinyl-tRNA. Theanticodon of selenocysteinyl-tRNA interacts with a stop codon in themRNA (UGA) instead of a serine codon. The selenocysteinyl-tRNA has aunique structure that is not recognized by the termination machinery andis brought into the ribosome by a dedicated specific elongation factor.An element in the 3′ non-translated region (UTR) of selenoprotein mRNAsdetermines whether UGA is read as a stop codon or as a selenocysteinecodon.

In some embodiments to the invention a cancer, e.g. CML, is classifiedaccording to responsiveness to a tyrosine kinase inhibitor, e.g. “classIII tyrosine kinase receptors”, which refers to a subclass of receptortyrosine kinases (RTKs). Imatinib is exemplary as an inhibitor. Theclass III RTKs, which include PDGFRa, PDGFRb, c-Fms, c-Kit and Fms-liketyrosine kinase 3 (Flt-3), are distinguished from other classes of RTKsin having five immunoglobulin-like domains within their extracellularbinding site as well as a 70-100 amino acid insert within the kinasedomain (Roskoski, R. (2005) Biochem. Biophys. Res. Commun. 338:1307-15).Structural similarities among class III RTKs results in cross-reactivitywith respect to ligands, as evidenced in the case of imatinib blockingPDGFRa, PDGFRb, c-Fms, and c-Kit.

Platelet-derived growth factor receptors (PDGFR) include PDGFR-alpha(PDGFRa) and the PDGFR-beta (PDGFRβ) (Yu, J. et al, (2001) BiochemBiophys Res Commun. 282:697-700). The PDGF B-chain homodimer PDGF BBactivates both PDGFRα and PDGFRβ, and promotes proliferation, migrationand other cellular functions in fibroblast, smooth muscle and othercells. The PDGF-A chain homodimer PDGF AA activates PDGFRα only. PDGF-ABbinds PDGFRα with high-affinity and in the absence of PDGFRα can bind ata lower affinity (Seifert, R. A., et al, (1993), J Biol Chem.268(6):4473-80). Recently, additional PDGFR ligands have been identifiedincluding PDGF-CC and PDGF-DD. Fibroblasts and other mesenchymal cellsexpress fibroblast-growth factor receptor (FGFR) which mediates tissuerepair, wound healing, angiogenesis and other cellular functions.

Cells.

Cells for use in the assays of the invention can be an organism, atissue sample, including a biopsy sample, etc. The invention is suitablefor use with any cell type, including primary cells, biopsy tissue

Cell types that can find use in the subject invention include stem andprogenitor cells, e.g. embryonic stem cells, hematopoietic stem cells,mesenchymal stem cells, neural crest cells, etc., endothelial cells,muscle cells, myocardial, smooth and skeletal muscle cells, mesenchymalcells, epithelial cells; hematopoietic cells, such as lymphocytes,including T-cells, such as Th1 T cells, Th2 T cells, Th0 T cells,cytotoxic T cells; B cells, pre-B cells, etc.; monocytes; dendriticcells; neutrophils; and macrophages; natural killer cells; mast cells;etc.; adipocytes, cells involved with particular organs, such as thymus,endocrine glands, pancreas, brain, such as neurons, glia, astrocytes,dendrocytes, etc. and genetically modified cells thereof. Hematopoieticcells may be associated with inflammatory processes, autoimmunediseases, etc., endothelial cells, smooth muscle cells, myocardialcells, etc. may be associated with cardiovascular diseases; almost anytype of cell may be associated with neoplasias, such as sarcomas,carcinomas and lymphomas; liver diseases with hepatic cells; kidneydiseases with kidney cells; etc.

The cells may also be transformed or neoplastic cells of differenttypes, e.g. carcinomas of different cell origins, lymphomas of differentcell types, etc.

Tumors of interest for imaging include carcinomas, e.g. colon, prostate,breast, melanoma, ductal, endometrial, stomach, dysplastic oral mucosa,invasive oral cancer, non-small cell lung carcinoma, transitional andsquamous cell urinary carcinoma, etc.; neurological malignancies, e.g.neuroblastoma, gliomas, etc.; hematological malignancies, e.g. childhoodacute leukemia, non-Hodgkin's lymphomas, chronic lymphocytic leukemia,malignant cutaneous T-cells, mycosis fungoides, non-MF cutaneous T-celllymphoma, lymphomatoid papulosis, T-cell rich cutaneous lymphoidhyperplasia, bullous pemphigoid, discoid lupus erythematosus, lichenplanus, etc.; and the like.

Lysates.

The cells, which may be cells after exposure to an agent or condition ofinterest, are lysed prior to analysis. Methods of lysis are known in theart, including sonication, non-ionic surfactants, etc. Non-ionicsurfactants include the Triton™ family of detergents, e.g. Triton™ X-15;Triton™ X-35; Triton™ X-45; Triton™ X-100; Triton™ X-102; Triton™ X-114;Triton™ X-165, etc. Brij™ detergents are also similar in structure toTriton™ X detergents in that they have varying lengths ofpolyoxyethylene chains attached to a hydrophobic chain. The Tween™detergents are nondenaturing, nonionic detergents, which arepolyoxyethylene sorbitan esters of fatty acids. Tween™ 80 is derivedfrom oleic acid with a C₁₈ chain while Tween™ 20 is derived from lauricacid with a C₁₂ chain. The zwitterionic detergent, CHAPS, is asulfobetaine derivative of cholic acid. This zwitterionic detergent isuseful for membrane protein solubilization when protein activity isimportant. The surfactant is contacted with the cells for a period oftime sufficient to lyse the cells and remove additional adherent cellsfrom the system.

Methods of cellular fractionation are also known in the art. Subcellularfractionation consists of two major steps, disruption of the cellularorganization (lysis) and fractionation of the homogenate to separate thedifferent populations of organelles. Such a homogenate can then beresolved by differential centrifugation into several fractionscontaining mainly (1) nuclei, heavy mitochondria, cytoskeletal networks,and plasma membrane; (2) light mitochondria, lysosomes, and peroxisomes;(3) Golgi apparatus, endosomes and microsomes, and endoplasmic reticulum(ER); and (4) cytosol. Each population of organelles is characterized bysize, density, charge, and other properties on which the separationrelies.

The isoelectricly focused protein is bound to a specific binding member.The term “specific binding member” as used herein refers to a member ofa specific binding pair, i.e. two molecules, usually two differentmolecules, where one of the molecules through chemical or physical meansspecifically binds to the other molecule. The complementary members of aspecific binding pair are sometimes referred to as a ligand andreceptor; or receptor and counter-receptor.

Binding pairs of interest include antigen and antibody specific bindingpairs, peptide-MHC-antigen complexes and T cell receptor pairs, biotinand avidin or streptavidin; carbohydrates and lectins; complementarynucleotide sequences; peptide ligands and receptor; effector andreceptor molecules; hormones and hormone binding protein; enzymecofactors and enzymes; enzyme inhibitors and enzymes; and the like. Thespecific binding pairs may include analogs, derivatives and fragments ofthe original specific binding member. For example, an antibody directedto a protein antigen may also recognize peptide fragments, chemicallysynthesized peptidomimetics, labeled protein, derivatized protein, etc.so long as an epitope is present.

Immunological specific binding pairs include antigens and antigenspecific antibodies; and T cell antigen receptors, and their cognateMHC-peptide conjugates. Suitable antigens may be haptens, proteins,peptides, carbohydrates, etc. Recombinant DNA methods or peptidesynthesis may be used to produce chimeric, truncated, or single chainanalogs of either member of the binding pair, where chimeric proteinsmay provide mixture(s) or fragment(s) thereof, or a mixture of anantibody and other specific binding members. Antibodies and T cellreceptors may be monoclonal or polyclonal, and may be produced bytransgenic animals, immunized animals, immortalized human or animalB-cells, cells transfected with DNA vectors encoding the antibody or Tcell receptor, etc. The details of the preparation of antibodies andtheir suitability for use as specific binding members are well-known tothose skilled in the art.

The binding member may be directly or indirectly labeled with anoptically detectable label, usually a chemiluminescent label. Ofinterest as a label are fluorophores. Fluorescence is a physicalphenomenon based upon the ability of some molecules to absorb and emitlight. With some molecules, the absorption of light at specifiedwavelengths is followed by the emission of light from the molecule of alonger wavelength and at a lower energy state. Such emissions are calledfluorescence and the emission lifetime is said to be the average periodof time the molecule remains in an excited energy state before it emitslight of the longer wavelength. Substances that release significantamounts of fluorescent light are termed “fluorophores”. This broad classincludes fluorescein isothiocyanate (FITC), fluorescein di-galactose(FDG); lissamine, rhodamine, Texas Red, phycoerythrin, allophycocyanin,6-carboxyfluorescein (6-FAM),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (6-JOE),6-carboxy-X-rhodamine (6-ROX),6-carboxy-2,4,4′,5′,7,7′-hexachlorofluorescein (6-HEX),5-carboxyfluorescein (5-FAM) or N,N,N,N-tetramethyl-6-carboxyrhodamine(6-TAMRA); dansyl chloride; naphthylamine sulfonic acids such as1-anilino-8-naphthalene sulfonic acid (“ANS”) and2-p-toluidinylnaphthalene-6-sulfonic acid (“TNS”) and their derivatives;acridine orange; proflavin; ethidium bromide; quinacrine chloride; andthe like.

The specific binding partner will bind with a cellular parameter ofinterest. Parameters may include a variety of post-translationalmodifications, e.g. phosphoserine, phosphotyrosine; acyl groups, etc. Inaddition to, or in combination with, a parameter can be any cellcomponent or cell product including receptor, protein or conformationalor posttranslational modification thereof, lipid, carbohydrate, organicor inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portionderived from such a cell component or combinations thereof. Parametersmay provide a quantitative readout, in some instances asemi-quantitative or qualitative result.

Parameters of interest include detection of cytoplasmic biomolecules,frequently biopolymers, e.g. polypeptides, polysaccharides,polynucleotides, lipids, etc. In one embodiment, parameters includespecific epitopes. Epitopes are frequently identified using specificmonoclonal antibodies or receptor probes. A parameter may be defined bya specific monoclonal antibody or a ligand or receptor bindingdeterminant.

Of interest for the methods of the invention are cells before, afterand/or during exposure to an agent or agents of interest. Candidatebiologically active agents may encompass numerous chemical classes,primarily organic molecules, which may include organometallic molecules,inorganic molecules, genetic sequences, etc. An important aspect of theinvention is to evaluate candidate drugs, select therapeutic antibodiesand protein-based therapeutics, with preferred biological responsefunctions. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, frequently at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules, including peptides, polynucleotides,saccharides, fatty acids, steroids, purines, pyrimidines, derivatives,structural analogs or combinations thereof.

Included are pharmacologically active drugs, genetic agents, etc.Compounds of interest include chemotherapeutic agents, anti-inflammatoryagents, hormones or hormone antagonists, ion channel modifiers, andneuroactive agents. Exemplary of pharmaceutical agents suitable for thisinvention are those described in, “The Pharmacological Basis ofTherapeutics,” Goodman and Gilman, McGraw-Hill, New York, N.Y., (1996),Ninth edition, under the sections: Drugs Acting at Synaptic andNeuroeffector Junctional Sites; Drugs Acting on the Central NervousSystem; Autacoids: Drug Therapy of Inflammation; Water, Salts and Ions;Drugs Affecting Renal Function and Electrolyte Metabolism;Cardiovascular Drugs; Drugs Affecting Gastrointestinal Function; DrugsAffecting Uterine Motility; Chemotherapy of Parasitic Infections;Chemotherapy of Microbial Diseases; Chemotherapy of Neoplastic Diseases;Drugs Used for Immunosuppression; Drugs Acting on Blood-Forming organs;Hormones and Hormone Antagonists; Vitamins, Dermatology; and Toxicology,all incorporated herein by reference. Also included are toxins, andbiological and chemical warfare agents, for example see Somani, S. M.(Ed.), “Chemical Warfare Agents,” Academic Press, New York, 1992).

Methods

A patient lesion is analyzed through any the methods described above.The tissue samples are analyzed for the presence of variations inproteomic biomarkers, e.g. changes in the total amount, or as a percentof total proteins of a particular isoform, which isoform may bedifferentially phosphorylated, glycosylated, prenylated, etc. Asdescribed above, the analysis will utilize a high throughput system foranalysis. The sample may be compared to samples from other regions ofthe lesion, to earlier time points in the disease, to normal tissues ofsimilar type, and the like.

The information about an individual patient from the analysis is usefulin therapeutic uses, where therapy can be adjusted to utilize theoptimal molecular targets, and can then be monitored over time.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the subject invention, and are not intended to limit thescope of what is regarded as the invention. Efforts have been made toensure accuracy with respect to the numbers used (e.g. amounts,temperature, concentrations, etc.) but some experimental errors anddeviations should be allowed for. Unless otherwise indicated, parts areparts by weight, molecular weight is average molecular weight,temperature is in degrees centigrade; and pressure is at or nearatmospheric.

Example 1

In order to accurately measure oncoprotein expression and activation inlimited clinical specimens, we have developed a nano-fluidic proteomicimmunoassay detection method (NIA) that combines isoelectric proteinfocusing and antibody detection. Here we demonstrate that we can usethis new technique to quantitate oncoprotein expression andphosphorylation in clinical specimens to precisely measure specificchanges in phospho-isomers of oncoproteins in vitro and in vivo.

Results

NIA Detection of Oncoprotein Expression in Clinical Specimens. NIAincorporates isoelectric focusing of proteins, followed by antibodydetection of specific epitopes with chemiluminescence. Thechemiluminescent signal is rendered as a chemiluminescenceisoelectropherogram (trace) of “relative luminescence units (RLU)” onthe y-axis vs. isoelectric point (pl) on the x-axis (FIG. 1a ). Aslittle as 2 picograms of MYC could be detected using 4 nanoliters ofrecombinant protein per capillary (final capillary concentration0.004-0.12 pg/nl) at the expected isoelectric point (pl) of 5.6 with alinear response (FIG. 1b , R²=0.9984). Concentrations of BCL2 could bedetected over up to a 3-log dynamic range at pl 6.7 (FIG. 1c ,R²=0.9647). Therefore, NIA is highly quantitative and sensitive over alarge dynamic range.

Next, we assessed the ability to detect oncoproteins in tumor cell linesboth in vitro and in vivo. Previously, we have described the generationof a conditional transgenic lymphoma model that uses the Tet-off systemto regulate oncoprotein expression. In our tumor derived cell lines, MYCand BCL2 were readily detected by NIA, comparable to Western analysis(FIG. 6). Each oncoprotein measurement from these tumor lysatesexhibited a characteristic peak profile defined by howpost-translational modifications influence its isoelectric point. Theprofile obtained from mouse tumor lines was different from therecombinant BCL2 described above because NIA is exquisitely sensitive tothe cationic charge associated with the histidine tag of the recombinantprotein. Our results elaborate how NIA can distinguish and measureoncoprotein expression from tumor cell lines.

To evaluate if we could quantitatively detect oncoprotein expression invivo, conditional mouse tumor-derived cell lines were inoculated intosyngeneic mice. Examination of serial tumor samples by fine needleaspiration (FNA) confirmed that both MYC and BCL2 expression could bereadily detected and quantitated in vivo by NIA with results comparableto Western analysis (FIG. 1d, e ). To measure oncoprotein expression inhuman tumors, MYC and BCL2 levels were measured in four lymphomas andtwo benign lymph nodes. High levels of MYC protein commonly associatedwith Burkitt's lymphoma and high levels of BCL2 characteristic offollicular lymphoma were detected (FIG. 2a ). Thus, NIA can reproduciblydetect changes in oncoprotein expression even from limited clinicalbiopsy specimens in mouse or human tumor specimens both in vitro and invivo.

Our results suggested that we may be able to precisely quantify levelsof oncoproteins in clinical specimens. Analysis was performed on a setof 27 human specimens that included Burkitt's lymphoma, follicularlymphoma, benign lymph nodes, or normal peripheral blood mononuclearcells (PBMC's) (set 1, FIG. 2b, c ). In our first series (set 1),Burkitt's lymphoma samples expressed MYC at a level higher than all ofthe other sample groups (Mann Whitney test, p<0.0001). All Burkitt'slymphoma specimens had a MYC level greater than 0.2 RLU, compared with11% of follicular lymphoma. Follicular lymphoma samples expressedsignificantly higher BCL2 than all other sample groups (Mann Whitneytest, p<0.0001); 89% of follicular lymphoma patients had a BCL2 levelhigher than 0.06 RLU, compared with none of the Burkitt's patients. BCL2was detectable and quantifiable even in normal specimens. NIA wasquantitative enough to distinguish statistically significant differencesin oncoprotein expression between lymphomas.

To validate our results, we next quantified MYC and BCL2 in a new seriesof clinical specimens including seven Burkitt's lymphoma, 11 follicularlymphoma, nine diffuse large B cell lymphoma (DLBCL) and five marginalzone lymphoma (MZ) specimens (FIG. 2b, c , set 2). We confirmed that theMYC threshold of 0.2 RLU and the BCL2 threshold of 0.06 RLU werestatistically significant for distinguishing Burkitt's from follicularlymphoma (Fisher's exact test, two tailed: p=0.0498 and 0.0474,respectively). We observed that two of nine DLBCL tumors overexpressedBCL2 and many tumors expressed high increased levels of MYC. Incontrast, marginal zone lymphomas expressed a low mean level of 0.18 RLUMYC. Hence, we found different levels of MYC and BCL2 expression in 44of 49 tumor specimens when compared with normal controls.

NIA Measurement of Oncoprotein Phosphorylation in Clinical Specimens. Animportant feature of NIA is that it can be used to identifyphosphorylated isoforms of a protein. ERK and distinct patterns ofphospho-isomers were detected in the normal and lymphoma specimens (FIG.7a ). Next, we identified MEK 1 and MEK 2 isomers (FIGS. 7b and 8).Experiments were performed in quadruplicate and were highlyreproducible. We could detect as little as a 10% difference inphosphorylated ERK and MEK. Our results show that NIA is highlysensitive, reproducible and quantitative.

Next, we evaluated if we could detect specific protein signaling changesin human tumors in response to targeted therapy. Treatment of the K562human CML cell line with imatinib in vitro resulted in changes inphosphorylation of STAT3/5, JNK, MEK1 and MEK2 and an expected increasein activated caspase-3 associated with apoptosis (FIG. 3, FIG. 9). Tofacilitate comparison with Western blot results, data was converted intoa “pseudo-blot” representation. In a pseudo-blot, the area under eachNIA peak is represented as a band at the corresponding isoelectricpoint. By visualizing different isoforms we were able to identify thatimatinib treatment was associated with a decrease in a specific ERK2phospho-isomer (FIG. 10a ). Thus, NIA appeared to detect a uniquesignaling change in response to the targeted therapeutic agent,imatinib.

To evaluate if we could identify changes in protein signaling in vivo inclinical specimens, we measured changes in oncoprotein signaling in thetumor cells in the peripheral blood of CML patients treated withtyrosine kinase inhibitors. All seven patients that responded totyrosine kinase inhibitor therapy exhibited a 54-100% decrease in amono-phosphorylated ERK2 isoform (note peak 3; FIG. 4a and FIGS. 10b, c), whereas two patients that were resistant to tyrosine kinase inhibitortherapy did not (FIG. 4c , table 1). Although basal ERK2 expression wasnot different (FIG. 11), the relative fold-change in ERK2phosphorylation was different between CML patients that respond versusthose that did not (unpaired t-test, 2 tailed=0.0007). Thus, NIA hasidentified a specific change in ERK phosphorylation that is associatedwith a clinical response to effective therapy.

Finally, we interrogated if NIA could be used to serially monitor theresponse of a patient's tumor to a potential biologic response modifierin vivo. Recently, we described that atorvastatin treatment causes tumorregression associated with specific changes in oncoprotein signaling ina mouse model of lymphoma. We investigated if atorvastatin hasbiological activity in human patients with lymphoma. We coulddemonstrate that patients treated with atorvastatin exhibited detectabledecreases in phosphorylation of STAT3/5 after eight days of treatment(FIG. 7). Thus, we found that atorvastatin has unanticipated in vivobiologic activity against human lymphoma cells.

We have developed the application of a highly sensitive and reproduciblenanoscale technology to measure oncoprotein expression andphosphorylation and to identify the biological response to therapeutics.Our method detected as little as 2 picograms of protein in as few as 4nanoliters of material over a 3-log dynamic range. We quantified MYC andBCL2 oncoproteins as well as changes in cancer signaling proteinsincluding ERK, MEK, JNK, STAT3/5 and apoptotic proteins such as caspase3. The requirement of only a small amount of material enabled us toanalyze serial samples of the same tumor in vivo, as we demonstrated inmouse models of lymphoma and in human tumor specimens; oncoproteinphosphorylation decreased in patient samples obtained before and afterinitiating treatment with imatinib or atorvastatin. We have demonstratedthat our method has utility both for pre-clinical and clinical studies.

Our method enables the analysis of solid tissue specimens such as fromtumor biopsies, as we illustrate through the analysis of lymphomaspecimens. Thus we could quantify differences in MYC and BCL2oncoprotein expression in normal lymph tissue and differentnon-Hodgkin's lymphoma subtypes, including Burkitt's and Follicularlymphomas. DLBCL and marginal zone lymphomas were not as readilydistinguished, underlining that lymphomas can have complex patterns ofoncoprotein overexpression and single proteomic measurements are notsufficient to distinguish most subtypes. The pathological diagnosis oflymphoma as well as other tumor subtypes requires the combination of anumber of analytical methods including histological, biochemical andgenetic techniques. Our approach has unique features that may complementthese existing methodologies.

We can precisely measure oncoprotein isoforms that are useful in theanalysis of the therapeutic response in clinical tumor specimens. Usingour approach, we were able to identify specific and rare ERK isoforms invivo in patients with CML that would only be detectable with largeamounts of tumor tissue for alternate techniques of 2-D blots or massspectrometry. Charge-based separation via isoelectric focusing of eachprotein isoform based upon its unique pl allowed simultaneousquantification of multiple isoforms of the same protein in relativeproportions that could not have been detected a priori by Western orFACS techniques. Our results would have been missed by conventionalimmunoassay or flow cytometric (FACS) techniques since commerciallyavailable immunoreagents for activated ERK are raised against the dualphospho-ERK epitopes. Unlike FACS, our samples did not requireprocessing into a single cell suspension, fixation or permeabilization.Furthermore, decreased ERK2 phosphorylation correlated with the clinicalresponse to TK inhibitors.

In addition, we were able to serially sample tumors in patients withlymphoma for a biologic response to a therapeutic agent, atorvastatin.Our observations illustrate that NIA can be used to interrogate theprotein signature of and identify biomarkers for response to known andnovel therapeutic agents.

Our results demonstrate how a nanoscale proteomic technology can be usedto uncover unanticipated changes in protein signaling in vivo. Smallperturbations in the equilibrium between active and inactive proteinisoforms may have dramatic effects on the biologic state of a cell. Ourapproach provides a new technique that can be incorporated intopre-clinical and clinical studies to evaluate subtle shifts in proteinabundance and modification that may be useful for the discovery of newdrugs that target protein pathways and the identification of biomarkersof therapeutic efficacy.

Methods

Nano-Fluidic Proteomic Immunoassay (NIA).

The NIA experiments were performed using a Firefly™ or CB1000™instrument (Cell Biosciences). Briefly, for each capillary analysis, 4nanoliters of 10 mg/ml lysate were diluted to 0.2 mg/ml in 200nanoliters HNG-based lysis buffer [20 mM Hepes pH 7.5, 25 mM NaCl, 0.1%,10% glycerol, Sigma Phosphatase Inhibitor Cocktail 1, CalbiochemProtease Inhibitor] or 200 nanoliters Bicine/CHAPS lysis buffer. 200nanoliters sample mix containing internal pl standards were added. TheFirefly system first performed a charge-based separation (isoelectricfocusing) in a 5 cm long, 100 micron inner-diameter capillary. Predictedpl's were calculated using Scansite. Each sample was run on a panel ofdifferent pH gradients (3-10, 5-9) to optimize the resolution ofdifferent peak patterns. After separation and photo-activatedin-capillary immobilization, proteins were detected with antibodies: MYCprotein expression was detected using the C-19 rabbit polyclonalantibody that recognizes mouse and human MYC (Santa Cruz Biotechnology).BCL2 protein expression was detected using the clone 124 mousemonoclonal antibody that recognizes human BCL2 (Dako Laboratories).Total ERK1/2 protein expression was detected using a rabbit polyclonalantibody that recognizes mouse and human ERK1/2 (Upstate). Phospho-ERK1/2 protein expression was detected using a mouse monoclonal antibodythat recognizes both mouse and human pERK 1/2 (Cell Signaling).Additional antibodies used were: activated caspase 3 (Cell Signaling),MEK1 (Upstate), pMEK1 (Novus), MEK2 (Abcam), pSTAT3 (Cell Signaling),pSTAT5 (Cell Signaling), pJNK (Cell Signaling), HSP70 (Novus).

NIA Peak Area Quantitation.

Quantitation of the peaks is performed using peak analysis software. Thestart and end of each peak, and a flat baseline were manually selected.The area of each peak was calculated by dropping verticals to thebaseline at the peak start and end, and summing the area between thestart and endpoints. NIA has been shown to be able to discriminatebetween and quantitate phosphorylated and unphosphorylated isoforms ofERK in a single sample, using a total ERK antibody. The areas underdifferent peaks within a single tracing represented various ERKisoforms. To calculate the percentage p-ERK2 peak 3, the area under peak3 was divided by the sum of the area under the total ERK peaks. Sampleswere run in duplicate.

NIA Pseudo-Blot Generation.

The pseudo-blots were created by a linear mapping of the signalintensity to a grayscale image. Each pseudo-blot lane is representativeof a single capillary and consists of horizontal bands correspondingproportionally to the signal present. Absence of signal is white, whileincreasing signal is seen as an increasing dark band. It should be notedthat what is seen as a single band of protein in a size-based western,can appear as a single band by NIA or multiple bands when multiplecharged isomers of the protein are present.

Human Tumor Samples.

Tissues were obtained and banked from patients per Stanford UniversityIRB-approved protocols. Informed consent was obtained from all subjects.Burkitt lymphomas and T-ALL tissues were frozen in OTC blocks.Follicular lymphomas were dissociated into single cell suspension andstored in heat inactivated FBS +10% DMSO in liquid nitrogen. CML and CLLcells were isolated from total blood by ficoll separation, resuspendedin heat inactivated FBS +10% DMSO, and stored in liquid nitrogen. Tumorcells stored in liquid nitrogen were thawed at 37° C. into pre-warmedPBS and washed once in PBS immediately before use.

Data and Statistical Analysis.

NIA Multipeak fitting and peak area calculations were done with Peak Fitv4.11 (Systat Software), using Gaussian peaks with variable widths, aspreviously described. To obtain R² correlation coefficients, the bestfit line was calculated by linear regression with IGOR Pro version 5.03(Wavemetrics). NIA quantitations of MYC and BCL2 were compared to therelative intensity of respective quantified Western Blot bands usingPearson correlation. Paired t-test (two-tailed) was used to analyzepercent of p-ERK2 peak 3 before and during treatment. Mann Whitney Ranksum test was performed on the panels of patient specimens using Prismv4.0 (GraphPad). Prism v4.0 (GraphPad) was also used for Fisher's exacttest (2 tailed) analysis of contingency table data for MYC and BCL2.

Normalization of NIA Data.

Normalization of NIA data was performed in a similar fashion astraditional western blots or other molecular biology assays. HSP70 wasused as a “housekeeping” protein for NIA normalization. Relativeluminescent units (RLU) were calculated by dividing the measured peakarea for protein of interest by the measured peak area of HSP-70 andexpressed as a percentage. Across different experiments, a standardizedlysate control was used for calibration of instruments and runs.

Recombinant Proteins.

MYC protein (Active Motif, Carlsbad, Calif.) or BCL2 protein (R&DSystems, Minneapolis, Minn.) were added to lysis buffer.

Tissue Culture.

Tumor derived cell lines were grown in RPMI (GIBCO) media containing 10%FCS, 1% penicillin/streptomycin (GIBCO), and 0.000003%betamercaptoethanol (Sigma). Cells were grown in T25 flasks inincubators maintained at 37 degrees with 5% carbon dioxide.

Imatinib Treatment In Vitro.

100 mg Imatinib (Novartis, Basel, Switzerland) tablets were dissolved insterile PBS at 37° C. and added to media for a final concentration of 10mM in vitro.

Conditional Oncogene Expression.

All experiments were performed with the approval from the StanfordUniversity Administrative Panel on Laboratory Animal Care. The TRE-MYCtransgenic line generated for these experiments was describedpreviously. The TRE-BCL2 transgenic lines generated for theseexperiments were generated in conjunction with R. Padua. The Eμ-tTAtransgenic line was kindly provided by H. Bujard₂. Mice were mated andscreened by PCR. Tumor derived cell lines were generated. Syngeneic micewere injected subcutaneously with lymphoma-derived cell lines containingtetregulatable MYC or BCL2. When tumors reached >1 cm₃, oncogeneexpression was suppressed in vivo by injecting mice with 100 μg ofdoxycycline in PBS IP and adding doxycycline (100 μg/ml) to the drinkingwater. In vitro, MYC or BCL2 oncogenes were inactivated in tumor derivedcell lines by the addition of doxycycline (0.01, 0.05, 2, or 20 ng/ml,final concentration) to the media.

Tumor Sampling of Transgenic Tumors by Fine Needle Aspiration (FNA).

We performed serial fine needle aspiration procedures (FNAs) on mice toobtain cell samples from subcutaneous lymphoma tumors before and threedays after oncogene inactivation. Continuous negative pressure wasapplied to a 2 ml syringe with 20 gauge needle while 10 passes were madethrough the subcutaneous tumor. Specimens were collected into PBS. Redblood cells were removed using Pharmalyse (BD). Each FNA procedureobtained an average of 7 million cells.

Western.

Western analysis was performed using conventional techniques. Lymphoidtissues were disrupted and protein was isolated in HNTG lysis buffer [20mM Hepes pH 7.5, 25 mM NaCl, 0.1%, 0.1% Triton X-100, 10% glycerol,Sigma Phosphatase Inhibitor Cocktail 1, Calbiochem Protease Inhibitor],then sonicated for 30 seconds, 5 minutes on ice (repeated twice). CMLcells and CML patient specimens were lysed in RIPA lysis buffer RIPAlysis buffer [25 mM Hepes pH 7.5, 150 mM NaCl, 1% NP 40, 0.25%Na-deoxycholate, 10% glycerol, Sigma Phosphatase Inhibitor Cocktail 1,Calbiochem Protease Inhibitor]. 50 ug protein was loaded in each lane,as quantitated by the Bicinchoninic Acid Protein Assay (Pierce).Proteins were electrophoresed on 10% Tris-HCl polyacrylamide gels at 100V for 60 min and transferred on PVDF membranes at 100 V for 60 min. Themembrane was blocked in 5% nonfat dry milk solution in TBS at 4-8° C.overnight. Blots were incubated with primary antibodies at 4-8° C.overnight. MYC, BCL2, ERK, p-ERK, activated caspase 3, pMEK1, pMEK1,pSTAT5, pJNK and HSP70 were detected with the same primary antibodiesused for NIA. Blots were washed three times with TBST and then incubatedfor one hour with secondary anti-mouse or anti-rabbit HRP-conjugatedantibodies (Amersham). ECL detection kit (Amersham) was used forantibody detection. The western data was quantitated using Image Quantand Data Acquisition & Analysis Version 7.3 software (Van Mierlo).

Phospho-Protein FACs.

Cells were fixed in 1.6% paraformaldehyde at 37° C. for 10 minutes,permeabilized with 100% methanol for 10 minutes at room temperature,then washed with PBS, centrifuged at 2000 RPM for 5 minutes,subsequently washed with PBS 1% BSA, then resuspended in 50 ul of PBS 1%BSA. 1 million cells per FACs tube were stained with 10 ulanti-Phospho-ERK1/2 (Thr202/Tyr204):PE (BD Biosciences Pharmingen) in100 ul PBS 1% BSA. After a 30 minute incubation in the dark, sampleswere washed once with PBS 1% BSA and subsequently analyzed using abenchtop FACSCAN (Becton-Dickinson) flow cytometer. 10,000 ungatedevents were collected per sample.

-   O'Neill, R. A., et al. Isoelectric focusing technology quantifies    protein signaling in 25 cells. Proc Nat/Acad Sci USA 103,    16153-16158 (2006)-   Felsher, D. W. & Bishop, J. M. Reversible tumorigenesis by MYC in    hematopoietic lineages. Mol Cell 4, 199-207 (1999).-   Kistner, A., et al. Doxycycline-mediated quantitative and    tissue-specific control of gene expression in transgenic mice. Proc    Natl Acad Sci USA 93, 10933-10938 (1996).-   Towbin, H., Staehelin, T. & Gordon, J. Electrophoretic Transfer of    Proteins from Polyacrylamide Gels to Nitrocellulose Sheets:    Procedure and Some Applications. PNAS 76, 4350-4354 (1979).-   O'Neill, R. A., et al. Isoelectric focusing technology quantifies    protein signaling in 25 cells. Proc Natl Acad Sci USA 103,    16153-16158 (2006).

Example 2 Nanoscale Quantification of Phosphorylated andUnphosphorylated ERK and MEK Isoforms Differentiates Tumor and Non-TumorClinical Specimens

We have developed the use of a highly sensitive microfluidicnano-immunoassay system (NIA) to perform detailed analysis of ERK andMEK activation in hematopoietic and solid tumors. We described above theuse of NIA for measurement of proteins in as little as 4 nL of lysatefrom lymphoma and leukemia specimens. Now, we present results measuringspecific isoforms of MAPK proteins in fine needle aspirates (FNAs) frompatients with solid tumors and in blood buffy coats from patients withMyelo-Dysplastic Syndrome (MDS).

Using a single antibody that recognizes both the phosphorylated andunphosphorylated isoforms of ERK, we can determine levels of each ERKisoform, and also percent phosphorylation of ERK. NIA revealed thatdifferent tumor types could be distinguished based upon differingpatterns of ERK isoforms. To determine if NIA can measure signalingchanges during treatment with novel drugs, human leukemic cells weretreated with a PLK-1 inhibitor, ON01910 (Onconova, Inc). Surprisingly,only 5% of ERK was phosphorylated in the TF1 cell line, whereas 90% ofMEK was phosphorylated. MEK2 phosphorylation decreased by 20% afterON01910 treatment. Our studies demonstrate NIA can be used to identifyspecific changes in the MAPK pathway that distinguish normal frommalignant cells and quantify response of cells to a targetedtherapeutic.

We determined optimal conditions for handling and processing of humansolid tumor FNA and blood buffy coat specimens for NIA proteomicanalysis; used NIA to measure phosphorylation and percentphosphorylation of MAPK proteins in clinical samples; determined ifphosphorylation and percent phosphorylation of MAPK proteins change upontargeted treatment; and evaluated proteomic response to novel therapiesin cells sampled from patients at different time points duringtreatment.

We found that NIA measurements are stable in specimens kept on ice up to60 minutes. Wth representative MEK measurements for FNA of solid tumorkept on ice for 0, 30, 60 minutes, statistical analysis indicated nosignificant difference between groups, corrected for multiple testing.See FIG. 14.

In particular, although AKT has been reported to be unstable, it wasfound that some AKT isoforms are stable for over 30 minutes and even 60minutes on ice. Therefore, optimal sample handling may include transporton ice. Removal of blood from a clinical sample can cause variability ofmeasurements, so desirable handling protocols do not remove blood. Itwas found that NIA measurements were the same in freshly lysed andfrozen samples.

The NIA results were validated with a mean coefficient of variance lessthan 5%, and the protocols were validated across 19 different tumortypes, including sarcoma, angiomyolipoma; dysgerminoma, testicular,melanoma, uterine, ovary, bladder, kidney, thyroid, adenoid cystic,neuroendocrine, thymus, adenocarcinoma, lung, colon, pancreas, stomach,squamous cell carcinoma, and head and neck. See FIG. 15.

ERK was found to be more highly phosphorylated in solid tumors than inlymphomas, using NIA measurements of percent ERK phosphorylation insolid tumor and lymphoma clinical specimens. Phosphorylated ERKdistinguishes tumor from non-tumor tissue, using NIA measurements of ERKin tumor (T) and non-tumor (N) clinical specimens. NIA analysis revealsincreased phosphorylation of ERK in some paired patient tumor andnon-tumor samples, decreased phosphorylation in others. NIA analysisalso revealed differential phosphorylation of ERK and MEK in MDS andcontrol patients.

Treatment with ON01910 altered phosphorylation of signaling proteins inTF1 cells. Using NIA measurements of phosphorylated JNK and MEK2 in TF1cells before and after treatment with ON01910, NIA analysis revealsincreased phosphorylation of JNK and decreased phosphorylation of MEK2in the human leukemic cell line TF1 after 24 hr of treatment withON01910.

Phosphorylation of STAT3/5 decreased in lymphoma patients treated withatorvastatin. Tumor cells were sampled from lymphoma patients day 1 andday 8 of atorvastatin treatment. Both NIA and FACS analysis demonstratethat CD20 positive cells from patients with lymphoma show a dramaticdecrease in STAT3 and STATS phosphorylation.

A 23-parameter protein profile was developed in solid tumors. The 23parameters were:

ERK Normalized Values: ERK Relative Ratios: Total ERK1/2 %phospho-ERK1/2 Phospho-ERK1/2 % Unphosphorylated ERK1/2 UnphosphorylatedERK1/2 % Phospho-ERK1 Total ERK1 % Unphosphorylated ERK1 Phospho-ERK1 %Phospho-ERK2 pERK1 % Unphosphorylated ERK2 ppERK1 % pERK1Unphosphorylated ERK1 % ppERK1 Total ERK2 % pERK2 Phospho-ERK2 % ppERK2pERK2 ppERK2 Unphosphorylated ERK2

The percent of phosphor-ERK is a particularly useful marker for kidneycancer. Each bar is a ratio of: the value of % phospho-ERK for thenon-tumor kidney specimen DIVIDED by the value of the % phospho-ERK forthe paired kidney tumor specimen. The Y-axis of the graph is on a logscale.

The percent of ppERK1 is a particularly selective marker for head andneck cancer.

NIA can be used to assess changes in total and phosphorylated proteinisoforms during therapeutic interventions in hematopoietic disorders andsolid tumors. FACS and NIA together allow measuring when proteomiceffects are specific to tumor cells and if novel agents preferentiallymodulate specific isoforms of signaling proteins.

Methods

Protocol for NIA analysis of Solid Tumor Fine Needle Aspirate (FNA).Cytopathologist performs at least 10 passes through tissue and collectsFNA into 4 mL RPMI. Put FNA suspension on ice for transport and storesat 4 C for pick up by lab. Minimize time between collection bycytopathologist and snap freezing FNA pellets. Divide FNA suspension into 1.5 mL tubes. Equally distribute suspension between 3 tubes. Spin 1.5mL tubes at 5000 rpm for 5 min at 4 C. Remove RPMI with P1000 filter tipvery carefully and avoid pellet as much as possible. Expect 1-2 μL maxresidual supernatant volume. Snap freeze pellets in liquid N2. Store at−80 C until ready for lysis. When lyse pellet, thaw on ice 1-2 min. Makeup MPER lysis buffer with following inhibitors: a. Aqueous ProteaseInhibitor (Cell Biosciences, 25×); b. Sigma Phosphatase Inhibitor (use1:100); c. Calbiochem Protease Inhibitor (use 1:500). Add 20 μL lysisbuffer to pellet approximately equivalent in size to 1 million cells.Completely resuspend cells in lysis buffer by pipetting up and down (cando a quick, low speed vortex). Leave on ice 30 min. Spin tube at maxspeed (14K rpm) for 10 min at 4 C. Remove supernatant with P200 filtertip. This is the lysate. Determine protein concentration of lysate usingPierce BCA kit. Optional step: Aliquot and snap freeze in liquid N2.Store −80 C. Calculate volume of lysate needed for final proteinconcentration of 0.1 mg/mL per well.

Load 20 μL primary antibodies per well and 30 μL secondary antibodiesper well in to 384-well plate and keep plate on ice at all times. Makeup luminol and load 30 μL per well in to plate. Make up Sample Diluent[MPER with DMSO Inhibitor Mix (50×)] and keep on ice. Aliquot 6 μLpremix per well to 0.5 mL tubes. Then add appropriate volume of SampleDiluent to tubes with premix. Add appropriate volume of lysate to tubeswith premix and Sample Diluent. Volume of Sample Diluent plus lysate isequal to 6 μL per well. Pipet up and down 20×, vortex at medium settingbriefly for 7 seconds, then pipet up and down 30×. Load 10 μL ofpremix/lysate per well. Spin plate 1000 rpm 10 sec to remove bubbles inwells.

Antibodies include ERK1/2 Millipore, 06-182 1:300 Goat anti-Rabbit,1:500; HSP70 Novus Biologicals, 600-571 1:5000 Goat anti-Mouse, 1:250;MEK1 Upstate, 07-641 1:100 Goat anti-Rabbit, 1:100; MEK2 Cell Signaling,9125 1:100 Goat anti-Rabbit, 1:100; pSTAT5 Upstate, 06-867 1:100 Goatanti-Rabbit, 1:100; pSTAT3 Abcam, AB30646 1:50 Goat anti-Rabbit, 1:100;AKT Santa Cruz, SC8312 1:50 Goat anti-Rabbit, 1:100.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing, for example, the compoundsand methodologies that are described in the publications which might beused in connection with the presently described invention. Thepublications discussed above and throughout the text are provided solelyfor their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior invention.

What is claimed is:
 1. A method for serial monitoring of specificchanges in protein isoforms in clinical tumor microbiopsy specimens, themethod comprising; performing nanofluidic proteomic immunoassay (NIA) ona tumor microbiopsy cellular sample from an individual for simultaneousquantification of multiple isoforms of a single protein, wherein saidcellular sample is maintained on ice for greater than 30 mins prior tocell lysis; determining the ratio of isoforms of the protein in saidcellular sample; comparing the ratio of isoforms in said cellular samplewith a paired sample from said individual, wherein the paired samplefrom said individual comprises two samples taken from the same tumor atdifferent time points; and identifying from the comparison of isoformratios in said paired samples a determination of specific changes inprotein isoforms, as measured by isoelectric focusing.
 2. The method ofclaim 1, wherein the microbiopsy cellular sample is less than 100,000cells.
 3. The method of claim 1, wherein the protein is an oncoprotein.4. The method of claim 3, wherein the isoform differs from alternativeisoforms of the same oncoprotein by total level of phosphorylation orpercent phosphorylation relative to the paired sample.
 5. The method ofclaim 4, wherein the oncoprotein is ERK2.
 6. The method of claim 1,wherein multiple time points from a single tumor in response totreatment are compared.
 7. The method of claim 1, wherein the cellularsample was previously frozen.
 8. The method of claim 1, wherein saidcellular sample comprises blood cells.
 9. The method of claim 1, whereinsaid microbiopsy cellular sample comprises kidney cancer cells.
 10. Themethod of claim 1, wherein said microbiopsy cellular sample compriseshead and neck cancer cells.
 11. The method of claim 1, wherein themicrobiopsy cellular sample is less than 1000 cells.
 12. The method ofclaim 1, wherein two or more oncoproteins are analyzed, and the two ormore oncoproteins are selected from pSTAT3, pSTAT5, myc, bcl2, MEK1,MEK2, pJNK, akt isoforms, total ERK1/2, phospho-ERK1/2, unphosphorylatedERK1/2, total ERK1, pERK1, ppERK1, unphosphorylated ERK1, total ERK2,pERK2, ppERK2, unphosphorylated ERK2; and ERK relative ratios for: %phospho-ERK1/2, % unphosphorylated ERK1/2, % phospho-ERK1, %unphosphorylated ERK1, % phospho-ERK2, % unphosphorylated ERK2, % pERK1,% ppERK1, % pERK2, % ppERK2, % phospho-MEK1 and % phospho-MEK2.
 13. Amethod for serial monitoring of specific changes in protein isoforms inclinical tumor microbiopsy specimens, the method comprising; performingnanofluidic proteomic immunoassay (NIA) on a tumor microbiopsy cellularsample of less than 100 cells from an individual for simultaneousquantification of multiple isoforms of a single protein, wherein saidcellular sample is maintained on ice for greater than 30 minutes priorto cell lysis; determining the ratio of isoforms of the protein in saidcellular sample; comparing the ratio of isoforms in said cellular samplewith a paired sample from said individual, wherein the paired samplefrom said individual comprises two samples taken from the same tumor atdifferent time points; and identifying from the comparison of isoformratios in said paired samples a determination of specific changes inprotein isoforms, as measured by isoelectric focusing.
 14. The method ofclaim 1 or 13, wherein said cellular sample is obtained by fine needleaspiration (FNA).
 15. A method for serial monitoring of specific changesin ERK2 protein isoforms in clinical CML microbiopsy specimens or bloodsamples to determine response to a tyrosine kinase inhibitor, the methodcomprising; performing nanofluidic proteomic immunoassay (NIA) on aclinical CML microbiopsy specimen or blood sample from an individual forsimultaneous quantification of multiple isoforms of a single proteinwherein said cellular sample is maintained on ice for greater than 30mins prior to cell lysis; determining the ratio of isoforms of theprotein in said clinical CML microbiopsy specimen or blood sample; andcomparing the ratio of isoforms in said clinical CML microbiopsyspecimen or blood sample with a paired sample from said individual,wherein the paired sample from said individual comprises two samplestaken at different time; and identifying from the comparison of isoformratios in said paired samples a determination of specific changes inprotein isoforms, as measured by isoelectric focusing, wherein thepercentage of the single phosphorylated form is down regulated in aclinical CML microbiopsy specimen or blood sample from a patient that isresponsive to the tyrosine kinase inhibitor.
 16. The method of claim 15,wherein the tyrosine kinase inhibitor is imatinib.